Environmental stressors transiently activate neural systems that inhibit pain responsiveness. This phenomenon, called stress-induced analgesia (SIA), engages both opioid and nonopioid mechanisms. We have recently reported that nonopioid SIA may be mediated by the mobilization of endogenous cannabinoid compounds, such as 2-arachidonoylglycerol (2-AG), in the midbrain periaqueductal gray (PAG) [Hohmann et al., Nature 435: 1108-1112, 2005]. Our results show, indeed, that nonopioid SIA is abolished by cannabinoid receptor antagonists, is associated with 2-AG accumulation in the PAG, and is enhanced by pharmacological agents that block 2-AG deactivation. However, the mechanisms governing 2-AG signaling in the PAG during SIA remain largely unknown. Our central hypothesis is that stress stimuli that result in nonopioid SIA activate phospholipase C (PLC) and diacylglycerol lipase (DGL) in select neurons of the PAG, causing the rapid mobilization of 2-AG. Newly released 2-AG engages local cannabinoid receptors to induce nonopioid SIA, and is subsequently deactivated through monoacylglycerol lipase (MGL)-mediated hydrolysis. Glutamatergic stimulation of group I metabotropic receptors (mGluR), which are positively coupled to PLC/DGL, may be responsible for initiating these events. We will test this hypothesis with a series of experiments, which are expected to demonstrate that: 1) foot shock stress elicits on-demand mobilization of 2-AG in the PAG by activating PLC/DGL;2) selective inhibition of MGL activity in the PAG increases 2-AG accumulation in this structure and amplifies SIA;3) group I mGluR activation in the PAG stimulates 2-AG mobilization through PLC/DGL to induce SIA;and 4) key components of the 2-AG signaling system (i.e. mGluRS, PLC, DGL, cannabinoid receptors, MGL) are expressed in select neurons of the PAG. The results of these studies will elucidate, for the first time, biochemical and physiological mechanisms governing 2-AG signaling in the brain and determine the role of this endocannabinoid mediator in pain modulation. Furthermore, the results will provide essential information on the mechanisms responsible for endocannabinoid deactivation in the brain, which may facilitate the development of novel analgesic and anti- stress medicines.